Perturbation of the T cell receptor (TCR)/CD3 complex by anti-receptor antibodies (Ab) mimcs antigen activation and stimulates an inositol phospholipid (InsPL) -specific phospholipase C (PLC) resulting in generation of second messengers. Of these metabolites, inositol 1,4,5)P4) control Ca2+ mobilization. This project addresses the role of intracellular Ca2+ in regulating this pathway. Murine T cells permeabilized with a bacterial lysin are used as a model. Intracellular free [Ca2+] is controlled by a calcium/magnesium/EGTA buffer. Aggregation of CD3 complexes with the combination of a specific monoclonal Ab (mAb) and a second Ab induced inositol phosphate accumulation in the absence of added Ca2+. CD3-induced InsPL hydrolysis increased with the free Ca2+ concentration reaching a maximum at 100-300 nM [Ca2+] and decrease thereafter. Increasing free [Ca2+] to 300 nM increased the overall efficiency of hydrolysis, without changing the affinities governing CD3 coupling to PLC. Permeabilized cell's response to CD3 aggregation diminished, due to a mixed type inhibition, when exposed to >300 nM free [Ca2+]. Ca2+ alone had no effect on inositol phosphate levels in permeabilized cells. Only polyphosphoinositides were cleaved, irrespective of the Ca2+ concentration. No accumulation of Ins(1)P/Ins(3)P was detected, indicating that direct hydrolysis of phosphatidylinositol did not occur. Free [Ca2+] above 300 nM shifted the relative levels of CD3-induced Ins(1,4,5) P3 and Ins (1,3,4,5) P4 accumulation in favor of the latter, and enhanced the conversion of authentic [3H] Ins (1,4,5)P3 to Ins (1,3,4,5)P4. These data suggest that, although free Ca2+ not requried, InsPL hydrolysis is optimally triggered by CD3 perturbation at intracellular Ca2+ levels approximating those observed in intact resting lymphocytes (100 nM). Ca2+ concentrations of a magnitude similar to those triggered in intact cells by InsPL-derived metabolites may negatively affect InsPL hydrolysis and promote Ins (1,3,4,5) P4 production, thus controlling the intracellular amounts of Ins (1,4,5)P3.